We have previously described a novel method for isolating murine hematopoietic stem cells capable of the reconstitution of lethally irradiated recipients, which depends solely upon dual wavelength FACS analysis of murine bone marrow cells stained with the fluorescent DNA-binding dye Hoechst 33342. This method, which appears to rely upon the differential ability of stem cells to efflux the Hoechst dye, defines an extremely small and homogeneous population of cells (termed SP cells) which is highly enriched for stem cells providing long term hematopoietic reconstitution. We show here that dual wavelength analysis of Hoechst dye stained human, rhesus, and miniature swine bone marrow cells reveals a small distinct population of cells which efflux the dye in a manner identical to murine SP cells. Like the murine SP cells, both human and rhesus SP cells are primarily CD34-negative and lineage marker-negative. In vitro culture studies demonstrate that rhesus SP cells are highly enriched for long term culture initiating cells (LTC-IC), an indicator of primitive hematopoietic cells, and have the capacity for differentiation into T cells. While rhesus SP cells do not initially possess any hematopoietic colony forming ability, they acquire to ability to form colonies after long term culture on bone marrow stroma, coincident with their conversion to a CD34 positive phenotype. These studies suggest the existence of a hitherto unrecognized population of hematopoietic stem cells which lack the CD34 surface marker classically associated with primitive human and nonhuman primate hematopoietic cells.